Name: catheterization of intestinal loops in ruminants
Uploaded: 2009-06-11T00:00:00
Description: Watch this Scientific Journal Video about Catheterization of Intestinal Loops in Ruminants at JoVE.com

在农业和农业食品加拿大研究中心，莱斯布里奇，阿尔伯达省，加拿大的许多人没有奉献精神和努力，这项研究可能已经完成。在这方面，我们感谢：塔拉谢尔顿前和术后护理的动物，手术准备，​​麻醉科，样品分析，并与视频影像的援助;兰迪王尔德和ESTELA哥斯达黎加前和术后护理;珍妮Gusse样品分析;拜伦李套管形象;和布赖恩Egland养羊。我们也感谢提供视频设备和意见，以及其他援助（即执行生产）布伦特Selinger（莱斯布里奇大学生物科学系），和课程的再开发中心，莱斯布里奇大学录像专长。这项研究是支持部分由高级食品材料网（GDI），加拿大阿尔伯塔省肉牛业发展基金（GDI），和加拿大农业和农业食品（GDI和JPK）的赠款。

实验过程： <ol><li>所有仪器蒸压;非autoclaveable材料Germex冷至少10分钟的灭菌水溶液消毒。 </li><li>羊acepromazine（0.05毫克/公斤），格隆（0.005毫克/公斤），与布托啡诺酒石酸盐（0.2毫克/公斤）IM的前药，并且留15-20分钟。 </li><li>剃光和颈部皮肤手术擦洗。一旦颈内静脉是可视化的，在静脉注射0.1毫升利多卡因资深大律师，引入静脉导管，并固定在手术过程中使用牲畜识别标签水泥和2“医院磁带皮肤...

<ol>
	<li>Vidal, J. E., McClane, B. A., Saputo, J., Parker, J., Uzal, F. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Effects+of+Clostridium+perfringens+beta-toxin+on+the+rabbit+small+intestine+and+colon.">Effects of Clostridium perfringens beta-toxin on the rabbit small intestine and colon.</a> Infect. Immun. 76, 4396-4404 (2008).</li><li>Santos, R. L., Zhang, S., Tsolis, R. M., Baumler, A. J., Adams, L. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Morphologic+and+molecular+characterization+of+Salmonella+typhimurium+infection+in+neonatal+calves.">Morphologic and molecular characterization of Salmonella typhimurium infection in neonatal calves.</a> Vet. Pathol. 39, 200-215 (2002).</li><li>Uzzau, S., Leori, G. S., Petruzzi, V., Watson, P. R., Schianchi, G., Bacciu, D., Mazzarello, V., Wallis, T. S., Rubino, S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Salmonella+enterica+serovar-host+specificity+does+not+correlate+with+the+magnitude+of+intestinal+invasion+in+sheep.">Salmonella enterica serovar-host specificity does not correlate with the magnitude of intestinal invasion in sheep.</a> Infect. Immun. 69, 3092-3099 (2001).</li><li>Gerdts, V., Uwiera, R. R. E., Mutwiri, G. K., Wilson, D. J., Bowersock, T., Kindane, A., Babiuk, L. A., Griebel, P. J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Multiple+intestinal+'loops'+provide+an+in+vivo+model+to+analyse+multiple+mucosal+immune+responses.">Multiple intestinal 'loops' provide an in vivo model to analyse multiple mucosal immune responses.</a> J. Immunol. Methods. 256, 19-33 (2001).</li><li>Greibel, P. J., Davis, W. C., Reynolds, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=An+analysis+of+the+growth+and+differentiation+of+b+cells+isolated+from+follicles+from+ileal+Peyer's+patch+of+sheep.">An analysis of the growth and differentiation of b cells isolated from follicles from ileal Peyer's patch of sheep.</a> Immunology. 75, 601-607 (1992).</li><li>Motyka, G., Greibel, P. J., Reynolds, J. D. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Agents+that+activate+protein+kinase+C+resue+sheep+ileal+Peyer's+patch+B+cells+from+apoptosis.">Agents that activate protein kinase C resue sheep ileal Peyer's patch B cells from apoptosis.</a> Eur. J. Immunol. 23, 1314-1321 (1993).</li></ol>

在此过程中，我们能够成功地建立在羊回肠内建立的肠袢的导管。坚持严格的术后护理，动物迅速恢复从程序和手术后恢复正常活动5-7天。我们决定建立循环内导管不影响肠黏膜免疫功能（数据未显示）和动物可以生活至少8周后手术。此过程允许我们引入不同的免疫车厢多次治疗，在延长的时间间隔，一旦动物有从手术中恢复，并进行实证衡量这些治疗的生理和免疫效果。 ?...

<table border="1"><tr><td> 分类/编号 </td><td> 公司 </td><td> 目录 </td></tr><tr><td colspan="3"> 外科用品 </td></tr><tr><td>外科手套</td><td> WDDC Å </td><td> 103091 </td></tr><tr><td>外科口罩</td><td> WDDC </td><td> MAX53013 </td></tr><tr><td>手术衣</td><td> CDMV b </td><td> 3365 </td></tr><tr><td> OB的围裙</td><td> WDDC </td><td> 900062 </td></tr><tr><td>手术帽</td><td> WDDC </td><td> 903271 </td></tr><tr><td> EZ刷子刷洗</td><td> WDDC </td><td> BDC377479 </td></tr><tr><td colspan="3"> 麻醉和药物 </td></tr><tr><td>麻醉机</td><td>博伊尔</td><td> </td></tr><tr><td> Sodasorb </td><td> WDDC </td><td> 108638 </td></tr><tr><td>喉镜</td><td> WDDC </td><td> </td></tr><tr><td>喉镜片（米勒刀片＃4） </td><td> WDDC </td><td> 902667 </td></tr><tr><td>气管插管（7.0 - 8.5毫米） </td><td> WDDC </td><td> </td></tr><tr><td>利多卡因气管内喷雾</td><td> WDDC </td><td> 103364 </td></tr><tr><td> Acepromazine </td><td> WDDC </td><td> AUS01020 </td></tr><tr><td>格隆</td><td> WDDC </td><td> SAB2880 </td></tr><tr><td>布托啡诺酒石酸</td><td> WDDC </td><td> AYE304541 </td></tr><tr><td>地西泮</td><td> WDDC </td><td> sab790 </td></tr><tr><td>硫喷妥钠</td><td> WDDC </td><td> 103508 </td></tr><tr><td>盐酸肾上腺素</td><td> WDDC </td><td> 103856 </td></tr><tr><td>多沙普仑</td><td>斯特拉思科纳药房</td><td> </td></tr><tr><td>异氟醚</td><td> WDDC </td><td> 100339 </td></tr><tr><td> Hemostam </td><td> WDDC </td><td> 103857 </td></tr><tr><td>氟尼辛</td><td> WDDC </td><td> sch140100 </td></tr><tr><td>恩诺沙星</td><td> WDDC </td><td> hll0049 </td></tr><tr><td>甲硝唑</td><td> WDDC </td><td> 101653 </td></tr><tr><td>氨苄青霉素</td><td> WDDC </td><td> NOV67185 - 6 </td></tr><tr><td>头孢唑啉</td><td> WDDC </td><td> nov69397 </td></tr><tr><td colspan="3"> 手术设备和用品 </td></tr><tr><td> Electroscapel </td><td>马坎</td><td> MV8 </td></tr><tr><td>加热垫</td><td>凯恩</td><td> PHMD - 48 </td></tr><tr><td>胃管（NALGENE管5 / 16“编号） </td><td> Fisher Scientific则</td><td> </td></tr><tr><td>针驱动程序</td><td> WDDC </td><td> 108338 </td></tr><tr><td>艾迪棕色forcep </td><td> WDDC </td><td> 911075 </td></tr><tr><td>大鼠牙齿forcep </td><td> CDMV </td><td> 5275 </td></tr><tr><td>凯利forcep </td><td> WDDC </td><td> 901133 </td></tr><tr><td>蚊子止血</td><td> WDDC </td><td> 108471 </td></tr><tr><td> Carmalt forcep </td><td> WDDC </td><td> 901225 </td></tr><tr><td>手术刀处理</td><td> WDDC </td><td> 900285 </td></tr><tr><td>手术刀刀片＃22 </td><td> WDDC </td><td> 900511 </td></tr><tr><td>梅岑鲍姆剪刀</td><td> WDDC </td><td> 901166 </td></tr><tr><td>夏普锋利的剪刀</td><td> WDDC </td><td> 108264 </td></tr><tr><td>巾钳</td><td> WDDC </td><td> 901213 </td></tr><tr><td>阿里斯组织forcep </td><td> WDDC </td><td> 901201 </td></tr><tr><td>老前辈的forcep </td><td> WDDC </td><td> 904226 </td></tr><tr><td>不锈钢碗</td><td> WDDC </td><td> 903601 </td></tr><tr><td>不锈钢套管（弯） </td><td>本地供应商</td><td> </td></tr><tr><td>导管导引</td><td> WDDC </td><td> 11440 </td></tr><tr><td>手术巾</td><td> CDMV </td><td> 7972 </td></tr><tr><td>一次性悬垂</td><td> WDDC </td><td> 900035 </td></tr><tr><td>无菌悬垂</td><td> CDMV </td><td> 2323 </td></tr><tr><td>披风（40 × 40厘米） </td><td> CDMV </td><td> 8358 </td></tr><tr><td>手术悬垂（7“× 11”开放） </td><td> CDMV </td><td> 269​​4 </td></tr><tr><td>薇乔（2-0） </td><td> WDDC </td><td> JJMJ333H </td></tr><tr><td>丝绸2-0 </td><td> WDDC </td><td> 105248 </td></tr><tr><td>羊肠线＃3 </td><td> WDDC </td><td> CSL547 - 2070 </td></tr><tr><td> Supramid＃1 </td><td> WDDC </td><td> CSL547 - 3040 </td></tr><tr><td>针（切割和非切割） </td><td> WDDC </td><td> </td></tr><tr><td colspan="3"> 导管 </td></tr><tr><td>硅橡胶管（0.040“的ID，0.085”外径） </td><td> Fisher Scientific则</td><td> 11 - 189 - 15D </td></tr><tr><td> Orbeseal软膏</td><td> WDDC </td><td> 106344 </td></tr><tr><td>清除硅（732多用途密封胶） </td><td>本地供应商</td><td> </td></tr><tr><td>钝年底针</td><td> WDDC </td><td> 905035 </td></tr><tr><td>标记（永久性的工业） </td><td>本地供应商</td><td> </td></tr><tr><td>导管袋（建造Elastoplast 7.5厘米） </td><td> WDDC </td><td> 104094 </td></tr><tr><td colspan="3"> 绷带 </td></tr><tr><td>医院磁带（2“） </td><td> WDDC </td><td> 103842 </td></tr><tr><td>医院磁带（1“） </td><td> WDDC </td><td> VLMRENF20 </td></tr><tr><td>纱布的4x4 </td><td> WDDC </td><td> 107551 </td></tr><tr><td>纱布抱住</td><td> WDDC </td><td> 107633 </td></tr><tr><td> Elastoplast7.5厘米</td><td> WDDC </td><td> 104094 </td></tr><tr><td> Elastoplast rippable绷带</td><td> WDDC </td><td> 104093 </td></tr><tr><td colspan="3"> 氖edles和注射器</td></tr><tr><td>注射器（60毫升） </td><td> WDDC </td><td> 104004 </td></tr><tr><td>注射器（10毫升） </td><td> WDDC </td><td> bdc301604 </td></tr><tr><td> 25G针</td><td> WDDC </td><td> BDC305122 </td></tr><tr><td> 20G针</td><td> WDDC </td><td> BDC305175 </td></tr><tr><td> 18G针</td><td> WDDC </td><td> 903353 </td></tr><tr><td colspan="3"> 静脉内 </td></tr><tr><td> Intracath静脉导管（16G 8“） </td><td> WDDC </td><td> bdc3831621 </td></tr><tr><td>静脉注射组（10滴） </td><td> WDDC </td><td> 101797 </td></tr><tr><td> 0.9％氯化钠溶液</td><td> WDDC </td><td> BAXJB1322P </td></tr><tr><td> Plasmalyte 148解决方案</td><td> WDDC </td><td> BAXJB2534 </td></tr><tr><td>葡萄糖</td><td> WDDC </td><td> MCT60190 </td></tr><tr><td>蠕动泵</td><td>科尔帕默</td><td> 7524-10 </td></tr><tr><td>牲畜识别标签水泥</td><td>本地供应商</td><td> </td></tr><tr><td colspan="3"> Sanatizers /消毒剂 </td></tr><tr><td> Germex解决方案</td><td> WDDC </td><td> mtc60270 </td></tr><tr><td>异丙醇</td><td> WDDC </td><td> 104590 </td></tr><tr><td> Stanhexidine洗面奶</td><td> WDDC </td><td> 109248 </td></tr><tr><td> Prepodyne解决方案</td><td> WDDC </td><td> 105710 </td></tr><tr><td> Prepodyne擦洗</td><td> WDDC </td><td> 105711 </td></tr></table> 西方的毒品分销中心有限公司，埃德蒙顿，AB 。 b的兽药经销中心公司，圣Hyacinthe，量化宽松政策。

catheterization of intestinal loops in ruminants

肠道是一个复杂的结构，不仅在营养物质的吸收，而且还充当之间的个人和外界的障碍。因此，肠道内起着举足轻重的作用，从肠道致病菌的免疫监视和保护。调查肠道生理学和免疫学通常采用“肠袢”作为实验模型。这些循环模型大部分都是非恢复手术过程中，研究短期（&lt;24小时）在肠道内的变化（1-3）。我们先前创建的恢复肠袢模型来具体衡量长期（&lt;6个月）羊暴露后疫苗，佐剂和病毒（4）肠道免疫变化。此过程的本地化处理，以具体的“循环”，让我们品尝这一领域的肠道。这种方法的一个显着的缺点是管理治疗的机会，即在手术时的单一窗口。此外，肠道黏膜和管腔内容的样本，只能采取在项目终止。上述模型中的其他显着的局限性，手术操作和必要的手术后的措施（如抗生素和止痛药的管理），可直接影响治疗本身和/或改变免疫功能，从而混淆的结果。因此，我们修改了我们的肠袢模型，通过长期导管插入循环。羊完全恢复的程序，并exteriorized导管的影响。值得注意的是，建立在循环导管，使我们能够引进在延长的时间间隔多次治疗后，手术和清理手术期间服用过的药物和手术后的时期的恢复。

Watch this Scientific Journal Video about Catheterization of Intestinal Loops in Ruminants at JoVE.com

Catheterization of Intestinal Loops in Ruminants

我们描述了一种新的手术方法catheterizing在羊回肠“肠袢”。一旦动物有从手术中恢复，并已清除抗生素和止痛药，多治疗，可以直接存入通过导管循环。

导尿在反刍动物的肠袢

The intestine is a complex structure that is involved not only in absorption of nutrients, but also acts as a barrier between the individual and the ...

catheterization-of-intestinal-loops-in-ruminants

Research

JoVE Journal

Biology

Investigating Intestinal Inflammation in DSS-induced Model of IBD

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease (CD). Both UC and CD, when present in the colon, generate a similar symptom profile which can include diarrhea, rectal bleeding, abdominal pain, and weight loss.1 Although the pathogenesis of IBD remains unknown, it is described as a multifactorial disease that involves both genetic and environmental components.2
There are numerous and variable animal models of colonic inflammation that resemble several features of IBD. Animal models of colitis range from those arising spontaneously in susceptible strains of certain species to those requiring administration of specific concentrations of colitis-inducing chemicals, such as dextran sulphate sodium (DSS). Chemical-induced models of gut inflammation are the most commonly used and best described models of IBD. Administration of DSS in drinking water produces acute or chronic colitis depending on the administration protocol.3 Animals given DSS exhibit weight loss and signs of loose stool or diarrhea, sometimes with evidence of rectal bleeding.4,5 Here, we describe the methods by which colitis development and the resulting inflammatory response can be characterized following administration of DSS. These methods include histological analysis of hematoxylin/eosin stained colon sections, measurement of pro-inflammatory cytokines, and determination of myeloperoxidase (MPO) activity, which can be used as a surrogate marker of inflammation.6
The extent of the inflammatory response in disease state can be assessed by the presence of clinical symptoms or by alteration in histology in mucosal tissue. Colonic histological damage is assessed by using a scoring system that considers loss of crypt architecture, inflammatory cell infiltration, muscle thickening, goblet cell depletion, and crypt abscess.7 Quantitatively, levels of pro-inflammatory cytokines with acute inflammatory properties, such as interleukin (IL)-1&beta;, IL-6 and tumour necrosis factor (TNF)-&alpha;,can be determined using conventional ELISA methods. In addition, MPO activity can be measured using a colorimetric assay and used as an index of inflammation.8
In experimental colitis, disease severity is often correlated with an increase in MPO activity and higher levels of pro-inflammatory cytokines. Colitis severity and inflammation-associated damage can be assessed by examining stool consistency and bleeding, in addition to assessing the histopathological state of the intestine using hematoxylin/eosin stained colonic tissue sections. Colonic tissue fragments can be used to determine MPO activity and cytokine production. Taken together, these measures can be used to evaluate the intestinal inflammatory response in animal models of experimental colitis.

Experimental models of inflammatory bowel disease have allowed us to examine the complex innate and adaptive immune responses associated with pathogenesis. Using histological scoring, quantification of pro-inflammatory cytokines and myeloperoxidase activity, one can begin to assess these responses seen in inflammatory bowel disease.

调查在DSS诱导的鸡传染性法氏囊病模型的肠道炎症

Inflammatory bowel disease (IBD) encompasses a range of intestinal pathologies, the most common of which are ulcerative colitis (UC) and Crohn's Disease ...

科研

生物学

A Video Protocol of Retroviral Infection in Primary Intestinal Organoid Culture

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding primary 3D epithelial structures in vitro. Both the architecture and physiological properties of these &#39;mini-guts&#39;, also called organoids, closely resemble their in vivo counterparts. This makes them an attractive model system for the small intestinal epithelium. Using retroviral transduction, functional genetics can now be performed by conditional gene overexpression or knockdown. This video demonstrates the procedure of organoid culture, the generation of retroviruses, and the retroviral transduction of organoids to assist phenotypic analysis of the small intestinal epithelium in vitro. This novel organotypic model system in combination with retroviral mediated gene expression provides a valuable tool for rapid analysis of gene function in vitro without the need of costly and time-consuming generation for transgenic animals.

This protocol explains primary Lgr5-positve organoid culture and the subsequent performance of retroviral transduction. This enables Cre-inducible overexpression or knockdown of the delivered transgene and allows functional studies to be carried out in the novel in vitro organotypic model system.

逆转录病毒感染的原发性肠化培养的视频协议

Lgr5-positive stem cells can be supplemented with the essential growth factors Egf, Noggin, and R-Spondin, which allows us to culture ever-expanding ...

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and composed of mature enterocytes, goblet cells and enteroendocrine cells; and crypts, residing proximal to the submucosa and the muscularis, harboring adult stem and progenitor cells and mature Paneth cells, as well as stromal and immune cells of the crypt microenvironment. Until the last few years, in vitro studies of small intestine was limited to cell lines derived from either benign or malignant tumors, and did not represent the physiology of normal intestinal epithelia and the influence of the microenvironment in which they reside. Here, we demonstrate a method adapted from Sato et al. (2009) for culturing primary mouse intestinal crypt organoids derived from C57BL/6 mice. In addition, we present the use of crypt organoid cultures to assay the crypt metabolic profile in real time by measurement of basal oxygen consumption, glycolytic rate, ATP production and respiratory capacity. Organoids maintain properties defined by their source and retain aspects of their metabolic adaptation reflected by oxygen consumption and extracellular acidification rates. Real time metabolic studies in this crypt organoid culture system are a powerful tool to study crypt organoid energy metabolism, and how it can be modulated by nutritional and pharmacological factors.

Real Time Analysis of Metabolic Profile in Ex Vivo Mouse Intestinal Crypt Organoid Cultures

Small intestinal crypt organoids cultured ex vivo provide a tissue culture system that recapitulates growth of crypts dependent on stem cells and their niche. We established a method to assay the metabolic profile in real time in primary mouse crypt organoids. We found organoids maintain physiological properties defined by their source.

代谢谱的实时分析<em&gt;离体</em&gt;鼠标肠隐窝类器官培养

The small intestinal mucosa exhibits a repetitive architecture organized into two fundamental structures: villi, projecting into the intestinal lumen and ...

A Protocol for Lentiviral Transduction and Downstream Analysis of Intestinal Organoids

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth factors. Since organoids are highly similar to the original tissue in terms of homeostatic stem cell differentiation, cell polarity and presence of all terminally differentiated cell types known to the adult intestinal epithelium, they serve as an essential resource in experimental research on the epithelium. The possibility to express transgenes or interfering RNA using lentiviral or retroviral vectors in organoids has increased opportunities for functional analysis of the intestinal epithelium and intestinal stem cells, surpassing traditional mouse transgenics in speed and cost. In the current video protocol we show how to utilize transduction of small intestinal organoids with lentiviral vectors illustrated by use of doxycylin inducible transgenes, or IPTG inducible short hairpin RNA for overexpression or gene knockdown. Furthermore, considering organoid culture yields minute cell counts that may even be reduced by experimental treatment, we explain how to process organoids for downstream analysis aimed at quantitative RT-PCR, RNA-microarray and immunohistochemistry. Techniques that enable transgene expression and gene knock down in intestinal organoids contribute to the research potential that these intestinal epithelial structures hold, establishing organoid culture as a new standard in cell culture.

In this video protocol we give a step by step explanation of lentiviral transduction in organoids of primary intestinal epithelium and of processing and downstream analysis of these cultures by quantitative RT-PCR, RNA-microarray and immunohistochemistry.

协议的慢病毒转导与肠组织体下游分析

Intestinal crypt-villus structures termed organoids, can be kept in sustained culture three dimensionally when supplemented with the appropriate growth ...

Measuring Pressure Volume Loops in the Mouse

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the mouse provides great potential to explore cardiac function at the molecular level. The mouse&#39;s small size does present some challenges in regards to performing detailed cardiac phenotyping. Miniaturization and other advancements in technology have made many methods of cardiac assessment possible in the mouse. Of these, the simultaneous collection of pressure and volume data provides a detailed picture of cardiac function that is not available through any other modality. Here a detailed procedure for the collection of pressure-volume loop data is described. Included is a discussion of the principles underlying the measurements and the potential sources of error. Anesthetic management and surgical approaches are discussed in great detail as they are both critical to obtaining high quality hemodynamic measurements. The principles of hemodynamic protocol development and relevant aspects of data analysis are also addressed.

This manuscript describes a detailed protocol for the collection of pressure-volume data from the mouse.

测量压力容量环中的鼠标

Understanding the causes and progression of heart disease presents a significant challenge to the biomedical community. The genetic flexibility of the ...

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and epithelial cell integrity, and disruption of the intestinal barrier function contributes to progression of gastrointestinal and systemic disease. Two simple methods are described here to measure the permeability of intestinal epithelium. In vitro, Caco-2BBe cells are plated in tissue culture wells as a monolayer and transepithelial electrical resistance (TER) can be measured by an epithelial (volt/ohm) meter. This method is convincing because of its user-friendly operation and repeatability. In vivo, mice are gavaged with 4 kDa fluorescein isothiocyanate (FITC)-dextran, and the FITC-dextran concentrations are measured in collected serum samples from mice to determine the epithelial permeability. Oral gavage provides an accurate dose, and therefore is the preferred method to measure the intestinal permeability in vivo. Taken together, these two methods can measure the permeability of the intestinal epithelium in vitro and in vivo, and hence be used to study the connection between diseases and barrier function.

In Vitro and In Vivo Approaches to Determine Intestinal Epithelial Cell Permeability

Two methods are presented here to determine intestinal barrier function. An epithelial meter (volt/ohm) is used for measurements of transepithelial electrical resistance of cultured epithelia directly in tissue culture wells. In mice, the FITC-dextran gavage method is used to determine the intestinal permeability in vivo.

肠上皮细胞通透性的体外及体内测定方法

The intestinal barrier defends against pathogenic microorganism and microbial toxin. Its function is regulated by tight junction permeability and ...

A Mouse Model of Intestinal Partial Obstruction

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including tumorous growths. However, the cellular remodeling mechanisms involved in, and caused by, intestinal obstructions are poorly understood. Several animal models of intestinal obstructions have been developed, but the mouse model is the most cost/time effective. The mouse model uses the surgical implantation of an intestinal partial obstruction (PO) that has a high mortality rate if it is not performed correctly. In addition, mice receiving PO surgery fail to develop hypertrophy if an appropriate blockade is not used or not properly placed. Here, we describe a detailed protocol for PO surgery which produces reliable and reproducible intestinal obstructions with a very low mortality rate. This protocol utilizes a surgically placed silicone ring that surrounds the ileum which partially blocks digestive movement in the small intestine. The partial blockage makes the intestine become dilated due to the halt of digestive movement. The dilation of the intestine induces smooth muscle hypertrophy on the oral side of the ring that progressively develops over 2 weeks until it causes death. The surgical PO mouse model offers an in vivo model of hypertrophic intestinal tissue useful for studying pathological changes of intestinal cells including smooth muscle cells (SMC), interstitial cells of Cajal (ICC), PDGFR&#945;+, and neuronal cells during the development of intestinal obstruction.

Intestinal obstructions are a partial or complete blockage of the intestine that can cause severe abdominal pain, nausea, vomiting, and preventing the passage of stool. This procedure for creating intestinal partial obsructions in mice is reliable in studying the mechanisms underlying pathological cell growth and death in the intestine.

小肠部分梗阻的小鼠模型

Intestinal obstructions, that impede or block peristaltic movement, can be caused by abdominal adhesions and most gastrointestinal (GI) diseases including ...

Intestinal Epithelial Regeneration in Response to Ionizing Irradiation

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the active proliferation of intestinal stem cells located at the bottom of intestinal crypts. However, during events of acute intestinal injury, these active intestinal stem cells undergo cell death. Gamma irradiation is a widely used colorectal cancer treatment, which, while therapeutically efficacious, has the side effect of depleting the active stem cell pool. Indeed, patients frequently experience gastrointestinal radiation syndrome while undergoing radiotherapy, in part due to active stem cell depletion. The loss of active intestinal stem cells in intestinal crypts activates a pool of typically quiescent reserve intestinal stem cells and induces dedifferentiation of secretory and enterocyte precursor cells. If not for these cells, the intestinal epithelium would lack the ability to recover from radiotherapy and other such major tissue insults. New advances in lineage-tracing technologies allow tracking of the activation, differentiation, and migration of cells during regeneration and have been successfully employed for studying this in the gut. This study aims to depict a method for the analysis of cells within the mouse intestinal epithelium following radiation injury.

The gastrointestinal tract is one of the most sensitive organs to injury upon radiotherapeutic cancer treatments. It is simultaneously an organ system with one of the highest regenerative capacities following such insults. The presented protocol describes an efficient method to study the regenerative capacity of the intestinal epithelium.

响应电离照射的肠上皮再生

The intestinal epithelium consists of a single layer of cells yet contains multiple types of terminally differentiated cells, which are generated by the ...

肠道炎症对已建立的小鼠结肠样的上皮影响

Studying the Epithelial Effects of Intestinal Inflammation In Vitro on Established Murine Colonoids

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic cell layer provides the first line of defense between microbial and immune populations and helps to modulate the intestinal immune response. Disruption of the epithelial barrier is a hallmark of inflammatory bowel disease (IBD) and is of interest for therapeutic targeting. The 3-dimensional colonoid culture system is an extremely useful in vitro model for studying intestinal stem cell dynamics and epithelial cell physiology in IBD pathogenesis. Ideally, establishing colonoids from the inflamed epithelial tissue of animals would be most beneficial in assessing the genetic and molecular influences on disease. However, we have shown that in vivo epithelial changes are not necessarily retained in colonoids established from mice with acute inflammation. To address this limitation, we have developed a protocol to treat colonoids with a cocktail of inflammatory mediators that are typically elevated during IBD. While this system can be applied ubiquitously to various culture conditions, this protocol emphasizes treatment on both differentiated colonoids and 2-dimensional monolayers derived from established colonoids. In a traditional culture setting, colonoids are enriched with intestinal stem cells, providing an ideal environment to study the stem cell niche. However, this system does not allow for an analysis of the features of intestinal physiology, such as barrier function. Further, traditional colonoids do not offer the opportunity to study the cellular response of terminally differentiated epithelial cells to proinflammatory stimuli. The methods presented here provide an alternative experimental framework to address these limitations. The 2-dimensional monolayer culture system also offers an opportunity for therapeutic drug screening ex vivo. This polarized layer of cells can be treated with inflammatory mediators on the basal side of the cell and concomitantly with putative therapeutics apically to determine their utility in IBD treatment.

作者聚焦：研究体外肠道炎症对已建立的小鼠结肠样蛋白的上皮效应  

We describe a protocol detailing the isolation of murine colonic crypts for the development of 3-dimensional colonoids. The established colonoids can then be terminally differentiated to reflect the cellular composition of the host epithelium prior to receiving an inflammatory challenge or being directed to establish an epithelial monolayer.

研究体 外 肠道炎症对已建立的小鼠结肠样的上皮影响

The intestinal epithelium plays an essential role in human health, providing a barrier between the host and the external environment. This highly dynamic ...